The long-term goal of our program is to advance the knowledge of RNA-ligand interactions and to apply the principles uncovered to the design, synthesis and discovery of new antiretroviral agents. The guiding hypothesis is that small molecules that target viral-specific RNA sites and prevent the formation of key RNA-protein and RNA-RNA complexes are promising candidates for antiretroviral drug discovery. The specific aims of this proposal are: (1) Develop rapid assays for the discovery of potent and specific viral RNA binders. To complement our Rev-RRE assay, we will develop a rapid assay for identifying high affinity and selective Tat-TAR inhibitors. We will also develop a fluorescence-based assay for the discovery of HIV DIS binders. (2) Design and synthesize RNA-selective ligands. We will conjugate "RNA-friendly" scaffolds (e.g., functionalized beta-and gamma-peptides, conformationally-constrained amino- and guanidine-glycosides) to sequence- and structure-selective elements (e.g., nucleobase analogs, RNA-selective intercalators, cis-Pt) to provide new families of potent and selective viral RNA binders. (3) Determine affinity and selectivity to HIV RNA sites. We will assess the Rev-RRE and Tat-TAR inhibitory activity as well as DIS affinity of all binders using the novel assays. (4) Evaluate anti HIV activity. Most promising binders will be evaluated for: (a) their ability to interfere with viral replication in HIV-1 infected cells, (b) their toxicity, and (c) their potential site(s) of interference. The rapid emergence of resistant viral strains necessitates the development of novel strategies for anti-HIV drug design. Small molecules that target pivotal viral RNA sites, and can specifically prevent the formation of key regulatory RNA-protein and RNA-RNA complexes, are proposed as promising candidates for antiretroviral drug development. Our integrated approach brings together organic synthesis, chemical diversity, novel solution and solid-phase assays, systematic structure-activity relationship studies, as well as in vivo anti-HIV tests, toxicity evaluation and mechanistic studies. It will yield new families of effective and specific binders that target key HIV-1 RNA sites. Potent and specific effectors of essential regulatory events will significantly advance the development of highly active antiretroviral agents.